PROJECT SUMMARY Dopaminergic projections from the midbrain to striatum regulate various cognitive and motor behaviors and are implicated in many psychiatric and neurodegenerative disorders. There are two anatomically and functionally distinct dopaminergic projections: from the substantia nigra pars compacta (SNc) to the caudate putamen (CPu) [the mesostriatal projection] and from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) [the mesolimbic projection]. The mesostriatal projection is critical for motor functions, while the mesolimbic projection is involved in drug addiction and emotional behavior. However, the molecular mechanisms under- lying the establishment of the specific dopaminergic synaptic connections in the striatum are unknown. We hypothesize that target (CPu or NAc)-specific molecules play crucial roles in the establishment of synapses from the two distinct dopaminergic projections. Using midbrain cultures, we found that CPu extract specifically induces presynaptic differentiation in SNc neurons, while NAc extract in VTA neurons. Partial purification indicated that the active molecules are 10?30 kDa. A microarray screen identified molecules that are differen- tially expressed between the CPu and NAc during dopaminergic synapse formation. Among them are BMP/ TGF? family members (which are 10?30 kDa): BMP2 and BMP6 are highly expressed in the CPu, while BMP3 and TGF?2 in the NAc. In situ hybridization and RT-PCR experiments confirmed their selective expressions in the CPu/NAc. In cultured midbrain neurons, these factors can induce dopaminergic presynaptic differentiation in a projection specific manner. Conversely, BMPR/TGF?R inhibitors blocked the effects of CPu/NAc extracts on dopaminergic presynaptic differentiation. Furthermore, in vivo knockdown of these BMP/TGF? resulted in region-specific (CPu or NAc) defects in dopaminergic synapse formation. Finally, application of BMP2 and TGF?2 to midbrain cultures activated distinct Smads, and distinct Smads were necessary for CPu and NAc extracts-dependent dopaminergic presynaptic differentiation. We propose that target-specific BMP/TGF? regulate differentiation of specific dopaminergic synaptic connections through the activation of distinct Smads. To test this hypothesis, we propose to: Aim 1: Determine whether BMP/TGF? contribute to dopaminergic axon targeting and/or synapse formation. Aim 2: Examine the physiological and behavioral consequences of BMP/ TGF? inactivation in the striatum. Aim 3: Investigate the role of specific Smads in BMP/TGF?-mediated dopaminergic synapse formation in vitro. Aim 4: Identify the BMP receptors and Smads that are critical for projection-specific dopaminergic synapse formation in vivo. We will use molecular cellular biological, biochemical, histological, imaging, electrophysiological, and behavioral approaches to address these aims. It is anticipated that this study will reveal the molecular mechanisms by which projection-specific dopaminergic synaptic connections are established in the mammalian brain, and will provide novel strategies for prevention and treatment of disorders implicated in dopaminergic projections such as substance use and dependence.